Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel method using combined chemical and enzymatic reactions to allow the preparation of covalently cross-linked DNA duplexes has been described. The method can be used to specifically link two complementary bases of a DNA duplex containing all four natural bases. The modified nucleotide 9-(2-deoxy-5-O-triphospho-beta-D-ribofuranosyl)-N6,N6-ethano -2,6-diaminopurine (6edDTP) was prepared by total chemical synthesis and was found to be incorporated into DNA duplexes in the place of 2'-deoxyguanosine 5'-O-triphosphate by the Klenow fragment of Escherichia coli DNA polymerase I, T4 and T7 DNA polymerases, avian myeloma virus reverse transcriptase, and rat DNA polymerase beta. Once incorporated, the aziridine of the nucleotide is rapidly opened by the N4 of the cytosine on the complementary strand to give cross-linked DNA, where the modified nucleotide is covalently joined to the complementary base by an ethano linkage. The duplexes produced were found to be recognized as substrates by various DNA polymerases. The Km for the incorporation of the 6edDTP into DNA catalyzed by the Klenow fragment of E. coli DNA polymerase I was found to be 29 microM, and the kcat was found to be 0.014 s-1. The modified nucleoside also served as a substrate for terminal deoxynucleotidyltransferase, where it was added to single-stranded DNA and then hybridized to a complementary strand, after which cross-linking of the two strands occurred within 1 min.
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PMID:A novel combined chemical-enzymatic synthesis of cross-linked DNA using a nucleoside triphosphate analogue. 198 67

Suramin, a polysulfonated naphthylurea widely used in the treatment of trypanosomiasis and onchocerciasis, is currently being investigated as an antitumor agent for the treatment of advanced cancer. Suramin exerts a wide variety of biological effects. We have shown that suramin inhibits cell proliferation and DNA synthesis in cultured HeLa cells. The replication in vitro of SV40 DNA is completely abolished by 40 microM suramin. The inhibition of DNA replication is due to inhibition of DNA polymerases alpha and delta, the replicative enzymes in eukaryotic cells. DNA polymerase alpha is sensitive to lower concentrations of suramin [concentration to achieve 50% inhibition (IC50) of 8 microM] than is DNA polymerase delta (IC50 36 microM), whereas DNA polymerase beta is relatively insensitive to the drug (IC50 of 90 microM). Suramin inhibits other replicative DNA polymerases such as Escherichia coli polymerase I (Klenow fragment) and Thermus aquaticus polymerase. Suramin is noncompetitive with both substrate deoxyribonucleotides and template-primers with respect to DNA polymerase inhibition. Much lower concentrations (8-30 microM) of the drug are required for 50% inhibition of DNA polymerases than for 50% inhibition of other enzymes such as protein kinase C and reverse transcriptase. These results show an important biological effect of this drug and indicate the need for more studies before its clinical use as an antitumor agent.
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PMID:Suramin affects DNA synthesis in HeLa cells by inhibition of DNA polymerases. 217 30

Four flavonoids (i.e., baicalein, quercetin, quercetagetin, and myricetin), known to be inhibitors of HIV-reverse transcriptase, have been shown to be more or less inhibitory to the activities of various cellular DNA and RNA polymerases. The degree of the inhibition varied depending on the combination of the flavonoid and the enzyme species: baicalein was moderately inhibitory to DNA polymerase gamma and E. coli DNA polymerase I; quercetin was strongly inhibitory to DNA polymerase beta and E. coli RNA polymerase and moderately inhibitory to DNA polymerase I; quercetagetin was a potent inhibitor for all of DNA polymerases alpha, beta, gamma, and I and RNA polymerase; myricetin was a strong inhibitor of DNA polymerases alpha and I and RNA polymerase. However, terminal deoxynucleotidyltransferase was virtually insensitive to inhibition by these flavonoids. The inhibition by the flavonoids was due to competition with the template.primer in the case of the DNA polymerases, whereas the inhibition was due to competition with the triphosphate substrate (GTP) in the case of RNA polymerase. The Ki values of these flavonoid inhibitors for DNA and RNA polymerases was determined.
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PMID:Mechanisms of inhibition of various cellular DNA and RNA polymerases by several flavonoids. 229 90

Compared to other T-lymphotropic human retroviruses, human T-cell leukemia (lymphotropic) virus I (HTLV-I) and HTLV-II, the acquired immunodeficiency syndrome (AIDS)-associated virus, HTLV-III, is a nontransforming cytopathic virus without immortalizing activity. Thus the virus replication is an important event in the manifestation of this disease, and the interruption of viral replication offers an important strategy for the control of AIDS. For this reason we have purified the reverse transcriptase (RT) from HTLV-III and from HTLV-III infected cells to study the structure-activity relationship of RT inhibitors developed in our laboratory. The cellular DNA polymerases from H9 cells were also purified to study the selectivity of RT inhibitors. Purified HTLV-III RT has several distinguishing features: (a) unlike the HTLV-I enzyme it is highly stable and can be kept for several weeks without any loss of activity; (b) using identical procedures of isolation the HTLV-III enzyme shows a much higher activity than does the enzyme from HTLV-I; (c) the Vmax for HTLV-III RT is by severalfold higher than that for the HTLV-I enzyme in the presence of (rC)n X (dG)12 and (rCm)n X (dG)12, and besides the usual template-primers used for RT assay this enzyme has a relatively high affinity for (rAm)n X (dT)12; and (d) the cationic requirements for the transcription of various template-primers are unusual. The purified enzyme has a molecular weight of 95,000-98,000, as judged by the gel filtration method. The purified HTLV-III RT was inhibited by a partially thiolated polycytidylic acid (5-mercaptopolycytidylic acid); the cellular DNA polymerase beta from H9 cells was not sensitive to 5-mercaptopolycytidylic acid. Germanin (synonym, suramin), an antiprotozoan drug, also inhibits HTLV-III RT activity, but the DNA polymerase alpha activity was also sensitive to Germanin. The nonspecific effect of Germanin is probably due to the high content of sulfonic acid residues. This paper describes new approaches for designing specific inhibitors of retroviral reverse transcriptases which may be useful in developing a potential drug against AIDS.
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PMID:Inhibitors of retroviral DNA polymerase: their implication in the treatment of AIDS. 241 Jan 12

dNTP(3'-OCH3), a 3'-O-methyl derivative of dNTP, is a chain terminator substrate for DNA synthesis catalyzed by AMV reverse transcriptase. The enzyme seems to be the only DNA polymerase susceptible to the inhibitor while all the other DNA polymerases tested are fully resistant to the nucleotide analog. The resistant polymerases are: E. coli DNA polymerase I, Klenow's fragment of DNA polymerase I, phage T4 DNA polymerase, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and calf thymus terminal deoxyribonucleotidyl transferase.
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PMID:3'-Hydroxymethyl 2'-deoxynucleoside 5'-triphosphates are inhibitors highly specific for reverse transcriptase. 242 65

The sugar-modified dTTP analogues 2',3'-didehydro-2',3'-dideoxy-thymidine 5'-triphosphate (ddeTTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), 3'-fluorothymidine 5'-triphosphate (FdTTP), and 3'-azidothymidine 5'-triphosphate (N3dTTP) are demonstrated to be very effective and selective inhibitors of the HIV-associated reverse transcriptase (HIV-RT). This conclusion is based on a comparison of the ID50 values of the compounds for the HIV-RT (ranging from 0.03 microM for ddeTTP to 0.1 microM for ddTTP) and the cellular DNA polymerase alpha (greater than 200 microM). DNA polymerase beta is partially affected by N3dTTP (ID50 = 31 microM) and by the other analogues (ID50 = 1-2.2 microM). FdTTP has proved as effective as N3dTTP (ID50 = 0.05 microM) in suppressing the HIV-RT activity. Kinetic analysis revealed for both dTTP analogues a competitive type of inhibition and the same K1 values (about 0.05 microM).
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PMID:Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5'-triphosphate in comparison to cellular DNA polymerases alpha and beta. 244 44

Ability of some new substrates containing the 5'-alpha-thiotriphosphate residue to terminate the DNA synthesis catalyzed by several DNA polymerases has been investigated. The cell-free test system contained the M13mp10 phage single-stranded DNA and a synthetic oligonucleotide primer. Reverse transcriptase from avian myeloblastosis virus catalyzed termination of DNA synthesis by 3'-azido-3'-fluoro- and 3'-amino-2',3'-dideoxythymidine-5'-(alpha-thio)triphosphates, whereas rat liver DNA polymerase beta and E. coli DNA polymerase I (Klenow's fragment) utilized only the second and the third compounds, and calf thymus DNA polymerase alpha failed to utilize any of the substrates. Low specificity of reverse transcriptase to different moieties of the substrate molecules is discussed.
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PMID:[Ability of 3'-substituted nucleoside phosphothioates to terminate DNA synthesis catalyzed by various DNA-polymerases]. 244 56

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66

An activity gel analysis was performed in order to examine the catalytically active component of human immunodeficiency virus (HIV) reverse transcriptase in purified enzyme preparations and HIV-infected cell extracts. Immunoaffinity purified HIV reverse transcriptase contains two proteins with molecular weights 66,000 and 51,000 in approximately equal proportions. After denaturing polyacrylamide gel electrophoresis and removal of sodium dodecyl sulfate, the p66 component of reverse transcriptase was sufficient for both DNA- and RNA-directed DNA synthesis. No DNA synthetic activity of p51 was observed. Recovery of p66 catalytic activity was approximately 10% that of DNA polymerase beta, and the density of the autoradiographic band corresponding to p66 was linear with enzyme concentration. No additional HIV-specific DNA polymerases besides p66 were observed in HIV-infected H9 cell extracts.
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PMID:Enzyme activity gel analysis of human immunodeficiency virus reverse transcriptase. 245 63

Several analogues of 2',3'-dideoxythymidine 5'-triphosphate [i.e., 3'-azido-2', 3'-dideoxythymidine 5'-triphosphate(Azdd TTP), 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate (ddeTTP), alpha, beta-methylene 3'-azido-2',3'-dideoxythymidine 5'-diphosphate, alpha, beta-methylene 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, and beta, gamma-methylene 3'-azido-2',3'-dideoxythymidine 5'-triphosphate] and 2',3'-didehydro-2',3'-dideoxycytidine 5'-triphosphate (ddeCTP) have been evaluated for their inhibitory effects on murine retroviral reverse transcriptase and various other DNA polymerases, including DNA polymerases alpha, beta, and gamma, terminal deoxynucleotidyl transferase, and DNA polymerase I. None of the compounds inhibited the activity of DNA polymerase alpha under the reaction conditions employed. When Mg2+ was replaced by Mn2+, however, DNA polymerase alpha was strongly inhibited only by ddeTTP. DNA polymerase beta activity was inhibited only by ddeTTP and ddeCTP. All the compounds, except for ddeCTP, inhibited DNA polymerase gamma activity, ddeTTP being a particularly strong inhibitor of gamma-polymerase (Ki = 3.5 nM). Terminal deoxynucleotidyl transferase was only slightly inhibited by any of the compounds. AzddTTP was a potent inhibitor of reverse transcriptase (Ki = 42 nM), but it also inhibited the activities of DNA polymerase gamma and DNA polymerase I.
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PMID:Differential inhibitory effects of several pyrimidine 2',3'-dideoxynucleoside 5'-triphosphates on the activities of reverse transcriptase and various cellular DNA polymerases. 247 Oct 54


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